Next Generation Sequencing for Food Authenticity ...Simplest NGS workflow for targeted sequencing...
Transcript of Next Generation Sequencing for Food Authenticity ...Simplest NGS workflow for targeted sequencing...
The world leader in serving science
Erin Dreyling, PhD
Regulatory Leader for Food Protection
Thermo Fisher ScientificMay 31, 2018
Next Generation Sequencing for Food Authenticity, Traceability and Safety
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The World Leader in Serving Science
Mission: We enable our customers to make the
world healthier, cleaner and safer.
Our Customer Focus:
We help accelerate innovation
and enhance productivity for
our customers.
in revenues$20B
employees>65,000
countries50
R&D investment>$900M
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The world leader in serving science
Ion Torrent
Sequencing
Technology
For Research Use Only. Not for use in diagnostic procedures.
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Simplest and Fastest NGS System for Targeted Sequencing
For Research Use Only. Not for use in diagnostic procedures.
Simplest NGS workflow for targeted sequencing
• <15 minutes to set up a sequencing run
• <45 minutes total hands-on time from DNA to data with Ion
Chef System
Fastest run time
• As little as 3.5 hours from sequence to BAM files
Lowest capital investment
• Single platform for all applications, with flexibility to scale
from 2 to 260 M reads per run*
Lowest DNA/RNA input requirements
• As little as 1 ng using Ion AmpliSeq technology
Easy setup and training
• Single-day installation, plug-and-play cartridge-based
reagents
Ion 520™ Chip5 M reads
Ion 530™ Chip15–20 M reads
Ion 540™ Chip60–80 M reads
Ion 510™ Chip2–3 M reads
Ion 550™ Chip100–130 M
reads
*Requires using two Ion 550 chips per run on Ion GeneStudio S5 Prime System to achieve >130 M reads per run
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• Ion torrent uses CMOS semiconductor technology just like
high end digital cameras
• Instead of light, ion torrent chip detects chemical changes as
the sequencing reaction proceeds
Ion Torrent Technology
High End Interchangeable Lens Camera CMOS Light Sensing Chip Ion Torrent Sequencing Chip
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• Why Targeted Sequencing?
• Focus on the genomic regions of
interest to minimize cost and
maximize speed
• Only 1.5% of the genome
encodes protein
• Many genes have now been
mapped to targets of interest
• Whole genome data creates
storage and analysis challenges
• Ion Ampliseq technology
enables researchers to focus
on the genomic regions they
care about
Targeted Sequencing
Internal Use Only / No external distribution
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~45 Minutes Hands-On Time from DNA to Data | Only 2 Pipetting Steps Per Sample
Automatedlibrary construction
Automated template
preparationSequencing
For Research Use Only. Not for use in diagnostic procedures.
Pipet
sample and primer
pools into Ion Chef
System for library prep
Load cartridge
into Ion Chef System
Pipet
library into Ion Chef
System for templating
and chip loading
Load cartridge
into Ion Chef System
Transfer chip
to Ion GeneStudio S5
System for sequencing
15 min
hands-on time
15 min
hands-on time
15 min
hands-on time
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Sequence Fast, Faster, and Faster with the Ion GeneStudio S5 Series
Ion GeneStudio S5
System
Ion GeneStudio S5 Plus
System
Ion GeneStudio S5 Prime
System
Speed*19 hrs 10 hrs 6.5 hrs
Output (max/day)**15 Gb 30 Gb 50 Gb
Number of reads (max/day)✝ 80 M 160 M 260 M
Compatible chips Ion 510, 520, 530, or 540 chips Ion 510, 520, 530, 540, and 550 chips
* Sequencing and analysis time based on Ion 540 chip.
** Requires using two Ion 540 chips per run on Ion GeneStudio S5 Plus System ✝Requires using two Ion 550 chips per run on Ion GeneStudio S5 Prime System
For Research Use Only. Not for use in diagnostic procedures.
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For multiplex samples,
multiple targets
Ion Torrent Applications: Food Integrity
Food Fraud, Authenticity,
Labeling
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Food Authenticity: where fingerprints are strong
We need to confirm whether a
species is present in sample
(e.g. allergies , religious taboos,
lower cost substance)
• Meat
• Halal Foods
• Plants, Spices
• GMO
Adulteration/Food Fraud
Mislabelling of fish, meat, fruit
and vegetable products is
common:
• Wild ↔ farmed
• Origin and feeding diet
• Organic versus inorganic
Mislabeling
We need to confirm the place of
origin of a product, label
declaration.
• Dairy
• Cheeses
• Plants, Spices
Authenticity, Origin
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Food Traceability by Ion Torrent™ sequencing
• Multi Species Identification
Accurate species ID by sequencing of cytochrome subunits and
other genes (“barcodes”) even in mixed samples
Sequencing results show the percentage composition of different
species in a sample
• Geographic Origin Determination of SNP frequencies by GBS of high diversity genes
• Pathogen Detection 16S gene sequencing for identification of bacterial organisms
18S/ITS gene sequencing for identification of fungal organisms
Metagenomics
Whole genome sequencing
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Thermo ScientificTM NGS solutions for Food Authenticity:
•The new Thermo Scientific™ Multi Species ID System is a complete,
automated, next generation sequencing workflow and software database for multi-
species ID to ensure confidence and consumer trust.
The NGS Food Integrity System includes:
Thermo Scientific™
Ion Chef™ System
Ion GeneStudio™
S5 System
Next Generation
Sequencer
Torrent Suite™ and
SGS ™ ALL Species
ID Analyses
Software
SGS™ ALL Species
ID DNA Analyser
Kits
DNA, Library & Template Preparation
34 hours
Sequencing
12 hrs
Data Analysis
1 hr
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NGS FOR FOOD AUTHENTICITY, TRACEABILITY AND SAFETY
In the present study an innovative NGS-based approach was tested for meat,
spices/cereals and fish species identification
In Partnership with:
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Origanum sp. Origanum
Tragelaphus strepsiceros Kudu
Felix catus Cat
Rattus norvegicus Rat
Vulpes vulpes Fox
Alces alces Elk
Coturnix japonica King quail
Bubalus bubalis Buffalo
Camelus dromedarius Camel
Crocodylus niloticus Crocodile
Lophura inornata Pheasant
Oryx leucoryx Oryx gazella
Alcelaphus buselaphus Gnu
Bos grunniens Cattle Yak
Equus asinus Donkey
Meles meles Badger
Tragelaphus scriptus Antilope
Corvus macrorhynchos Daw
Mustela erminea Weasel
Ondatra zibethicus Muskrat
Anas sp Mallard duck
Crocodylus siamensis Crocodile
Phasianus colchicus Pheasant
Alectoris chukar Partridge
Aepyceros melampus Impala
Origanum sp. Origanum
Ovis aries Sheep
Capra hircus Goat
Lepus capensis Hare
Oryctolagus cuniculus Rabbit
Macropus rufus Kangaroo
Capreolus capreolus Roe Deer
Cervus elaphus Red Deer
Rangifer tarandus Reindeer
Antidorcas marsupialis Springbok
Equus hemionus Zebra
Lama glama Lama
Gallus gallus Chicken
Canis familiaris Dog
Bison bison Bison
Cervus dama Fallow Deer
Equus caballus Horse
Sus scrofa Pork
Bos taurus Beef
Meleagris galopavo Turkey
Cairina moscata Duck
Alopochen aegptiacus Goose
Struthio camelus Ostrich
Columba livia Pigeon
Numida meleagris Guinea fowl
Dromaius novaehollandiae Emu
MEAT SPECIES RESULTS
• Number of samples tested: 148 Number of species tested: 49
• Species mix: up to 3 Spiked level: 1%, 10%, 50%
99.3
0.7
Meat Species
positve negative
95.3
4.7
Meat species spiked 1%
positive negative
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Origanum sp. Origanum
Laurus_nobilis Sweet bay
Manihot_esculenta Cassava
Mentha spicata Spearmint
Myristica fragrans Nutmeg
Ocimum basilicum Sweet basil
Oryza_sativa Rice
Panicum miliaceum Millet
Papaver somniferum Opium poppy
Petroselinum crispum Parsley
Pimpinella anisum Anis
Piper nigrum Black pepper
Rosmarinus_officinalis Rosemary
Sesamum indicum Sesame
Sinapis_alba White mustard
Sorghum_bicolor Sorghum
Thymus_vulgaris Garden Thyme
Triticum_aestivum Wheat
Triticum_durum Durum wheat
Zingiber_officinale Garden Ginger
Laurus_nobilis Sweet bay
Manihot_esculenta Cassava
Mentha spicata Spearmint
Myristica fragrans Nutmeg
Ocimum basilicum Sweet basil
Origanum sp. Origanum
Allium schoenoprasum Wild chives
Allium_sativum Garlic
Anethum graveolens Dill
Argemone sp Prickly poppy
Avena sativa Oat
Brassica napus Rape
Capsicum annuum Cayenne
pepper
Carum carvi Caraway
Ceratonia siliqua St John's bread
Conium maculatum Poison henlock
Coriandrum sativum Coriander
Crocus sativus Saffron
Cuminum_cyminum Cumin
Curcuma longa Turmeric
Elettaria cardamomum Cardamom
Foeniculum vulgare Sweet fennel
Glycine max Soybean
Hordeum vulgare Barley
Juniperus_communis Juniper
Origanum sp. Origanum
Allium schoenoprasum Wild chives
Allium_sativum Garlic
Anethum graveolens Dill
Argemone sp Prickly poppy
PLANTS SPECIES (SPICES/CEREALS) RESULTS
• Number of samples tested: 347 Number of species tested: 39
• Species mix: up to 5 Spiked level: 1%, 5%, 10%, 20%
98.9
Plant species
positive negative
97.1
2.9
Plant species spiked 1%
positive negative
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Origanum sp. Origanum
Trisopterus luscus Norway pout
Cynoglossus senegalensis Witch flounder
Oncorhynchus
chrysogaster
Pink salmon
Lophius piscatorius Angler
Oncorhynchus nerka Sockeye salmon
Pangasianodon
hypophthalmus
Silver carp
Scomber scombrus Atlantic mackerel
Oncorhynchus gorbuscha Pink salmon
Merluccius hubbsi Argentine hake
Merluccius productus North Pacific hake
Macruronus magellanicus Patagonian grenadier
Merluccius gayi South Pacific hake
Thunnus obesus Bigeye tuna
Origanum sp. Origanum
Salmo salar Atlantic Salmon
Thunnus albacares Yellowfin tuna
Gadus morhua Atlantic cod
Hippoglossus hippoglossus Pacific halibut
Limanda limanda Common dab
Merluccius merluccius European hake
Melanogrammus aeglefinus Haddock
Katsuwonus pelamis Skipjack tuna
Thunnus alalunga Albacore
Pleuronectes platessa European plaice
Molva molva Ling
Sander lucioperca Pike-perch
Pollachius pollachius Pollack
FISH SPECIES RESULTS
• Number of samples tested: 78 Number of species tested: 26
• Species mix: up to 2 Spiked level: 1%, 2%, 5%, 10%
94.4
5.6
Fish species >2% spiked
positive negative
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• The NGS workflow identified all of the species tested individually
• Identification of pure material at spiked levels higher than 5% was successfully
obtained for more than 94% of the samples tested
• At the level of 1% spiking, more that 95% of the samples were correctly assigned
to the species name
• When combined, meat, plants and fish species could be analyzed simultaneously
in a single NGS run which significantly reduces the NGS costs
• Extension of the databases is an on-going activity to include additional species
with various collaborators
Conclusions
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• Retailer asked for NGS
spices in Curry mixture
(containing more than 10
spices according to label)
• All the spices except
laurel (scientifica name
Laurus nobilis) could be
detected
• NGS spices was
performed and the
majority of the sample (>
90%) was composed by a
very similar plant of the
laurel family but not
Laurus nobilis (laurel) -
potentially a toxic plant
• The supplier was mixing
in the curry preparation
not Laurel but something
else similar to Laurel but
with less than 10% of true
laurel
Real-Time Response Case Study: Laurel raw material
Rapid ResponseHours to days to sequence sample, and
provide accurate analysis back
Customer SolutionSpecific for Plant Species ID testing in
food
SITUATION RESPONSE VALUE DELIVERED
Increased profitabilityFast and accurate analysis for prevention
of intentional adulteration of food supply
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• Retailer asked for grouper
identification in a fish fillet
that should be a single
grouper species. Therefore
no NGS was as performed
because the sample was a
fillet (single species
product)
• Identification through
Sanger sequencing that is
appropriate for single
species products originated
a mixture of DNA
sequences and no
identification results
• NGS was suggested to
the retailer
• NGS revealed 4 different
fish species and some of
them not included on the
list of authorized fish
species to be
commercialized – one of
the species toxic
• Supplier after knowing the
NGS results confirmed
that in spite of the fillet
format of the product it
was not a true fillet but
processed fish sample
with a fillet format
Real-Time Response Case Study: Grouper fillet
Rapid ResponseHours to days to sequence sample, and
provide accurate analysis back
Customer SolutionSpecific for Fish Species ID testing in
food
Scientific ExpertiseHighly skilled and supportive scientists
dedicated to food safety and integrity
SITUATION RESPONSE VALUE DELIVERED
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Appendix
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Ion Torrent Technology
• Ion torrent chips contain
millions of microscopic wells
• DNA is copied onto beads,
then loaded into wells
• DNA on bead is single
stranded, sequential flow of
nucleotides over chip builds
complement strand
• Each extension releases H+,
measured per well and
translated into sequence
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Plug-and-Play Reagents on Ion GeneStudio S5 Series Systems
For Research Use Only. Not for use in diagnostic procedures.
Large HD
touch
screen
Temperature-controlled
chip clamp
Sequencing reagent
cartridgeWash solution and
waste cartridge(behind W2 solution)
Automated
cleaning
solution
Standard power
Gas
Water
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• Output: DNA sequences
• Each DNA molecule is sequenced (ATCGTGTA…..)
• Millions of DNA molecules are sequenced individually and simultaneously
• Detect all different DNA in a sample
• Provides identification when compared with sequence databases
• Thousands of species are included in the databases
• Identifies content species in food products
• Untargeted approach
DNA Barcodes for Food Authenticity